PURIFICATION OF THE KPNI DNA METHYLTRANSFERASE AND PHOTOLABELING OF THE ENZYME WITH S-ADENOSYL-L-METHIONINE

An Escherichia coli strain overproducing the KpnI DNA methyltransferas e (M . KpnI) was constructed by cloning the kpnlM gene downstream from the inducible T7 phage Phi 10 promoter. A method involving three chro matographic steps has been developed to purify M . KpnI to homogeneity . The purified enzyme has a pH optimum around 7.3 and is inhibited by salts. M . KpnI can be photolabeled by UV-irradiation of the enzyme in the presence of S-adenosyl-L-[methyl-H-3]methionine ([methyl-H-3]AdoM et). Photolabeling results from a specific interaction between M . Kpn I and AdoMet, as indicated by the dependence of photolabeling on nativ e enzyme conformation and by the inhibitory effect of the AdoMet analo gs, sinefungin and S-adenosyl-L-homocysteine (AdoHcy).